Epistatic mutations in PUMA BH3 drive an alternate binding mode to potently and selectively inhibit anti-apoptotic Bfl-1

Elife. 2017 Jun 8;6:e25541. doi: 10.7554/eLife.25541.


Overexpression of anti-apoptotic Bcl-2 family proteins contributes to cancer progression and confers resistance to chemotherapy. Small molecules that target Bcl-2 are used in the clinic to treat leukemia, but tight and selective inhibitors are not available for Bcl-2 paralog Bfl-1. Guided by computational analysis, we designed variants of the native BH3 motif PUMA that are > 150-fold selective for Bfl-1 binding. The designed peptides potently trigger disruption of the mitochondrial outer membrane in cells dependent on Bfl-1, but not in cells dependent on other anti-apoptotic homologs. High-resolution crystal structures show that designed peptide FS2 binds Bfl-1 in a shifted geometry, relative to PUMA and other binding partners, due to a set of epistatic mutations. FS2 modified with an electrophile reacts with a cysteine near the peptide-binding groove to augment specificity. Designed Bfl-1 binders provide reagents for cellular profiling and leads for developing enhanced and cell-permeable peptide or small-molecule inhibitors.

Keywords: apoptosis; binding specificity; biochemistry; biophysics; human; protein-protein interaction; structural biology.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Apoptosis Regulatory Proteins / chemistry
  • Apoptosis Regulatory Proteins / genetics*
  • Apoptosis Regulatory Proteins / metabolism
  • Crystallography, X-Ray
  • Minor Histocompatibility Antigens
  • Models, Molecular
  • Mutation*
  • Peptide Fragments / chemistry
  • Peptide Fragments / genetics*
  • Peptide Fragments / metabolism
  • Protein Binding
  • Proto-Oncogene Proteins / chemistry
  • Proto-Oncogene Proteins / genetics*
  • Proto-Oncogene Proteins / metabolism
  • Proto-Oncogene Proteins c-bcl-2 / antagonists & inhibitors*


  • Apoptosis Regulatory Proteins
  • BBC3 protein, human
  • BCL2-related protein A1
  • Bax protein (53-86)
  • Minor Histocompatibility Antigens
  • Peptide Fragments
  • Proto-Oncogene Proteins
  • Proto-Oncogene Proteins c-bcl-2